I use 3T3-L1 fat cells. After it differentiated into mature adipocytes, I first starved the cells with serum-free starvation medium for two hours, and then stimulated the cells with insulin at concentrations of 0, 1nM, 10nM, and 100nM. The wb results showed that the wells with the insulin value of 0 p -akt still has a very strong signal, this signal should theoretically be 0. Has anyone encountered this situation? Where does the problem occur?
Hello Jia Qz ,
There may be several reason.
The media which you are using is not appropriate or the duration Of starvation .
Here I am suggesting some points which you can consider…
1. Use serum free media because serum have some amount of insulin.
2. If not getting the result, than replace the media with KRH Buffer containing BSA.
3.Check the temporal effect of Starvation like at the interval of 30, 60,90,120,180 minutes etc by examination of pAkT.
4. Check your antibody, may be it is detecting AkT, not p-AKT.
Hopefully, these points will help you to overcome the situation.
Thank You
There are two reasons why the p-AKT signal appears without insulin. One, the cells were not starved because serum contains large quantities of Insulin-like Growth Factor ( IGF) that can phosphorylate Akt just like insulin. So do starve cells in serum-free DMEM for 2 -3 hours before the actual insulin stimulation. Second possibility is that you are using advanced DMEM to starve cells. Advanced DMEM contains insulin and, therefore, is not feasible for cell starvation.
Thanks
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